title: The bile acid receptor TGR5 does not interact with β-arrestins or traffic to endosomes but transmits sustained signals from plasma membrane rafts.
abstract: TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.
authors.affiliation: Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria 3052, Australia.
authors.forename: Dane D
authors.initials: DD
authors.lastname: Jensen
authors.forename: Cody B
authors.initials: CB
authors.lastname: Godfrey
authors.forename: Christian
authors.initials: C
authors.lastname: Niklas
authors.forename: Meritxell
authors.initials: M
authors.lastname: Canals
authors.forename: Martina
authors.initials: M
authors.lastname: Kocan
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Poole
authors.forename: Jane E
authors.initials: JE
authors.lastname: Murphy
authors.forename: Farzad
authors.initials: F
authors.lastname: Alemi
authors.forename: Graeme S
authors.initials: GS
authors.lastname: Cottrell
authors.forename: Christoph
authors.initials: C
authors.lastname: Korbmacher
authors.forename: Nevin A
authors.initials: NA
authors.lastname: Lambert
authors.forename: Nigel W
authors.initials: NW
authors.lastname: Bunnett
authors.forename: Carlos U
authors.initials: CU
authors.lastname: Corvera
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D019390:Arrestins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002756:Cholagogues and Choleretics / Q000494:pharmacology; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism; D003840:Deoxycholic Acid / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D009828:Oleanolic Acid / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011799:Quinazolines / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D013876:Thiophenes / Q000494:pharmacology; D020032:Tyrphostins / Q000494:pharmacology; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; D000970:Antineoplastic Agents; D019390:Arrestins; D002756:Cholagogues and Choleretics; D004791:Enzyme Inhibitors; C473772:GPBAR1 protein, human; D011799:Quinazolines; D043562:Receptors, G-Protein-Coupled; D013876:Thiophenes; D020032:Tyrphostins; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D003840:Deoxycholic Acid; C101044:RTKI cpd; D010649:Phenylalanine; D009828:Oleanolic Acid; C080985:batimastat; D000242:Cyclic AMP; C512478:EGFR protein, human; D066246:ErbB Receptors; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
keywords: Arrestin; Bile Acid; Endocytosis; G Protein-coupled Receptors (GPCR); Lipid Raft